Biomedical subjects

H Kropshofer

Publications and source records attributed to H Kropshofer.

35 records · Page 2Linked to original sources

PubMed1996

Kinetic analysis of peptide loading onto HLA-DR molecules mediated by HLA-DM.

The nonclassical major histocompatibility complex class II molecule HLA-DM (DM) has recently been shown to play a central role in the class II-associated antigen presentation pathway: DM releases invariant chain-derived CLIP peptides (class II-associated invariant chain protein peptide) from HLA-DR (DR) molecules and thereby facilitates loading with antigenic peptides. Some observations have led to the suggestion that DM acts in a catalytic manner, but so far direct proof is missing. Here, we investigated in vitro the kinetics of exchange of endogenously bound CLIP for various peptides on DR1 and DR2a molecules: we found that in the presence of DM the peptide loading process follows Michaelis-Menten kinetics with turnover numbers of 3-12 DR molecules per minute per DM molecule, and with KM values of 500-1000 nM. In addition, surface plasmon resonance measurements showed that DM interacts efficiently with DR-CLIP complexes but only weakly with DR-peptide complexes isolated from DM-positive cells. Taken together, our data provide evidence that DM functions as an enzyme-like catalyst of peptide exchange and favors the generation of long-lived DR-peptide complexes that are no longer substrates for DM.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1996

Affinity enhancement of a recombinant antibody: formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion.

In antigen-antibody interactions, the high avidity of antibodies depends on the affinity and number of the individual binding sites. To develop artificial antibodies with multiple valency, we have fused the single-chain antibody Fv fragments to core streptavidin. The resulting fusion protein, termed scFv::strep, was found after expression in Escherichia coli in periplasmic inclusion bodies. After purification of the recombinant product by immobilized metal affinity chromatography, refolding and size-exclusion FPLC, tetrameric complexes resembling those of mature streptavidin were formed. The purified tetrameric scFv::strep complexes demonstrated both antigen- and biotin-binding activity, were stable over a wide range of pH and did not dissociate at high temperatures (up to 70 degrees C). Surface plasmon resonance measurements in a BIAlite system showed that the pure scFv::strep tetramers bound immobilized antigen very tightly and no dissociation was measurable. The association rate constant for scFv::strep tetramers was higher than those for scFv monomers and dimers. This was also reflected in the apparent constants, which was found to be 35 times higher for pure scFv::strep tetramers than monomeric single-chain antibodies. We could also show that most of biotin binding sites were accessible and not blocked by biotinylated E.coli proteins or free biotin from the medium. These sites should therefore facilitate the construction of bispecific multivalent antibodies by the addition of biotinylated ligands.

Antibodies↗

PubMed1995

Self-release of CLIP in peptide loading of HLA-DR molecules.

The assembly and transport of major histocompatibility complex (MHC) class II molecules require interaction with the invariant chain. A fragment of the invariant chain, CLIP, occupies the peptide-binding groove of the class II molecule. At endosomal pH, the binding of CLIP to human MHC class II HLA-DR molecules was counteracted by its amino-terminal segment (residues 81 to 89), which facilitated rapid release. The CLIP (81-89) fragment also catalyzed the release of CLIP(90-105) and a subset of other self-peptides, probably by transient interaction with an effector site outside the groove. Thus, CLIP may facilitate peptide loading through an allosteric release mechanism.

Amino Acid Sequence↗

PubMed1995

Interference of distinct invariant chain regions with superantigen contact area and antigenic peptide binding groove of HLA-DR.

In the endoplasmic reticulum, MHC class II alpha beta dimers associate with the trimeric invariant chain (li), generating a nine-subunit (alpha beta li)3 complex. In the presence of li, the peptide binding groove is blocked, so that loading with self or antigenic peptides can only occur after proteolytic removal of li in specialized post-Golgi compartments. The class II-associated invariant chain peptide region of li (about residues 81-104) is known to mediate binding to class II molecules and blockade of the groove, but this does not exclude additional contact sites for li. Using a set of overlapping li peptides and recombinant soluble li, we demonstrate here that a large segment of li encompassing approximately residues 71 to 128 interacts with HLA-DR molecules. The N- and C-terminal regions of this li segment appear to bind outside the peptide groove to the contact area for the staphylococcal superantigen Staphylococcus aureus enterotoxin B on the alpha 1 domain. The core region of this segment (residues 95-108) prevents binding of antigenic peptides, probably by interaction with the peptide groove. Occupation of the groove with antigenic peptides abolishes binding not only of the core region, but also that of those li peptides that bind outside the groove. These findings suggest the existence of distinct conformational states of class II molecules, with li binding preferentially to one form.

Amino Acid Sequence↗

PubMed1995

Structural features of the invariant chain fragment CLIP controlling rapid release from HLA-DR molecules and inhibition of peptide binding.

The invariant chain (Ii) prevents binding of ligands to major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum and during intracellular transport. Stepwise removal of the Ii in a trans-Golgi compartment renders MHC class II molecules accessible for peptide loading, with CLIP (class II-associated Ii peptides) as the final fragment to be released. Here we show that CLIP can be subdivided into distinct functional regions. The C-terminal segment (residues 92-105) of the CLIP-(81-105) fragment mediates inhibition of self- and antigenic peptide binding to HLA-DR2 molecules. In contrast, the N-terminal segment CLIP-(81-98) binds to the Staphylococcus aureus enterotoxin B contact site outside the peptide-binding groove on the alpha 1 domain and does not interfere with peptide binding. Its functional significance appears to lie in the contribution to CLIP removal: the dissociation of CLIP-(81-105) is characterized by a fast off-rate, which is accelerated at endosomal pH, whereas in the absence of the N-terminal CLIP-(81-91), the off-rate of C-terminal CLIP-(92-105) is slow and remains unaltered at low pH. Mechanistically, the N-terminal segment of CLIP seems to prevent tight interactions of CLIP side chains with specificity pockets in the peptide-binding groove that normally occurs during maturation of long-lived class II-peptide complexes.

Amino Acid Sequence↗

PubMed1994

Ligand motifs of HLA-DRB50101 and DRB11501 molecules delineated from self-peptides.

Antigenic peptides are presented to CD4+ T cells by MHC class II molecules via a highly polymorphic peptide-binding groove. The two HLA-DR alleles isotypically expressed on HLA-DR15Dw2-positive cells, DRB1*1501 (DR2b) and DRB5*0101 (DR2a) molecules, show a number of differences in polymorphic residues of the beta-chain, including the Gly-Val-dimorphism at position beta 86. Therefore, different requirements for interaction of peptides with these alleles must be expected. In this study, naturally processed self-peptides were eluted from purified HLA-DR15Dw2 molecules and related to DRB1*1501 or DRB5*0101 molecules by binding assays. An alignment of self-peptides and foreign peptides allowed the delineation of putative anchor motifs. N- and C-terminally truncated and alanine-substituted derivatives of the DR15Dw2 restricted myelin basic protein epitope MBP(85-105) confirmed their validity. Thus, DRB5*0101 requires a bulky hydrophobic residue (F or Y) at position i as a primary anchor, and Q or an aliphatic residue, such as V, I, or M, at position i + 3; positively charged residues at positions i + 7 and i + 8 are secondary anchors. For DRB1*1501, a nonaromatic, hydrophobic anchor (L, V, or I) at position i is supplemented by a bulky hydrophobic residue (F or Y) at position i + 3 as primary anchor; an additional hydrophobic side chain represented by M, I, V, or F occurs at position i + 6. Therefore, MBP(85-105) seems to contain two MHC interaction sites for DRB1*1501 and DRB5*0101, respectively, that may contribute to its immunodominance. Because HLA-DR15 Dw2 is associated with susceptibility to develop multiple sclerosis, the delineation of ligand motifs of the two DR2 alleles may help to study the interaction between potential autoantigenic peptides and these molecules in the future.

Alleles↗

PubMed1994

Association of HLA and multiple sclerosis.

Multiple Sclerosis (MS) is a demyelinating disease of the central nervous system. Although the cause of MS is still unknown, it is considered an autoimmune disease based on the composition of inflammatory infiltrates in the brain and on parallels with a T-cell-mediated animal model of demyelinating diseases called experimental allergic encephalomyelitis (EAE). Similar to other autoimmune diseases, the immunogenetic background, in particular the MHC/HLA type, contributes to susceptibility. This review summarizes the current knowledge about the association between HLA background and MS as well as immunological findings in EAE and MS. Finally, an attempt is made to explain how structural interactions between disease-associated HLA type and binding of an autoantigenic peptide could relate to disease.

Alleles↗

PubMed1993

Self-peptides from four HLA-DR alleles share hydrophobic anchor residues near the NH2-terminal including proline as a stop signal for trimming.

Naturally processed MHC class II-associated peptides proved to be heterogeneous in size, varying from 13 to 25 amino acids. Truncation variants suggested sequence motifs that afford the amino termini to be shifted for obtaining an alignment: a 9- to 11-residue core region that is bordered by primary anchor residues is surrounded by extra sequences of variable lengths and hitherto unknown functions. Herein we present bulk sequencing analyses of self-peptides from four HLA-DR alleles and HLA-DQw7 clearly showing that the length of most of the NH2-terminal preanchor sequence is limited to 1 to 3 residues. Most strikingly, proline is the dominant residue reappearing at positions 2 and 3 in any allele. Proline revealed to function as a stop signal for NH2-terminal trimming as well as a secondary anchor: crude cytosolic and endosomal peptide fractions could be processed by aminopeptidases in vitro, whereupon DR1 binding peptides with increased affinity were generated. In addition, aminopeptidase treatment of DR1: self-peptide complexes implied that proline together with sterical constraints of the MHC molecule do protect the peptides' NH2-termini from further processing, whereas their COOH-termini were accessible to cathepsin B processing. Finally, bulk sequencing profiles contained signals from further putative anchor residues clustering in the NH2-terminal region:tyrosine, phenylalanine, leucine, isoleucine, and valine are enriched at positions 2 to 4 in DR1, DR5, and DR6, however, at positions 4 to 6 in DR3. Isotype-specificity is demonstrated by DQw7 displaying glutamine and asparagine at position 2. Obviously, the degenerate occurrence of aromatic or aliphatic side chains close to the NH2-terminal guarantees for essential interactions with a hydrophobic pocket of the investigated DR molecules. Most probably, this pocket is located in the nonpolymorphic DR alpha-chain rationalizing previous findings of promiscuous peptide binding to different DR alleles.

Alleles↗

PubMed1993

Modulation of p145c-kit function in cells of patients with acute myeloblastic leukemia.

The function of the steel factor receptor, p145c-kit, in patient-derived acute myeloblastic leukemia (AML) cells was investigated. Steel factor stimulation of AML cells coexpressing p145c-kit and the progenitor cell antigen CD34 resulted in complete receptor down-regulation, a marked decrease of CD34 antigen expression, and the induction of the granulocyte lineage antigen CD15. These changes in surface marker expression paralleled morphological differentiation to granulated blasts and promyelocytes. Interestingly, the same phenotype was achieved by IL-3 stimulation of AML cells. p145c-kit extracellular domain-specific antibodies had either blocking or enhancing effects on ligand binding, receptor phosphorylation and down-regulation, and induction of cell proliferation. Correlations of these phenomena with distinct effects of antibody stimulation on cell substrate phosphorylation provide clues for the dissection of the p145c-kit signal and the analysis of its relevance for AML.

Animals↗

PubMed1993

Evidence for cobinding of self- and allopeptides to human class II major histocompatibility antigen DR1 by energy transfer.

Purified human class II major histocompatibility antigen HLA-DR1 was subjected to high-performance gel filtration with fluorescence detection to investigate simultaneous binding of two classes of peptides: the N-terminally fluoresceinated allopeptides fluorescein isothiocyanate (FITC)-conjugated DR1 beta-(66-78) and FITC-conjugated DR3 beta-(66-78), derived from the third hypervariable region of the beta chain of DR1 and DR3, respectively, and the DR1-associated self-peptide SP3, carrying the fluorophor 7-amino-4-methyl-coumarin-3-acetic acid (AMCA) at the N terminus. By analyzing the dimer-associated fluorescence signals, we measured an interpeptide energy transfer AMCA-->FITC that proved to be peptide-specific: it did not occur after replacement of the allopeptide by the DR1-restricted peptide IM-(18-29) from influenza matrix protein, whereas it was restored by SP3, due to the high homology of SP3 and allopeptide. Transfer analyses with truncated AMCA-SP3 and AMCA-IM-(18-29) are consistent with Leu-3 being a common anchor residue of both peptides that allows an interaction with the hydrophobic specifity pocket around Ala-37 of the alpha 1 domain. This interaction is mirrored by the intrinsic fluorescence of neighboring Trp-43: we found the protein-peptide transfer Trp(DR1)-->AMCA with AMCA-SP3 but with none of the allopeptides. Since each energy transfer affords close proximity of two fluorophors, the following picture emerges: self- or foreign peptides bind to the DR1 binding cleft by occupation of previously described specificity pockets. Simultaneously, allopeptides of the third hypervariable region or homologous peptides may occupy a cryptic binding site by displacing the beta 1-helix that normally lines the binding groove. Thus, the described complexes raise additional possibilities for the molecular basis of auto- or alloreactivity.

Amino Acid Sequence↗

PubMed1993

Characterization of peptides bound to extracellular and intracellular HLA-DR1 molecules.

Exogenous antigens are internalized by antigen-processing cells and processed within vesicular compartments to produce antigenic peptides that bind to newly synthesized MHC II molecules. These MHC class II peptide complexes are displayed at the plasma membrane and stimulate specific CD4+ T cells. In the present study, we established a method to isolate intracellular MHC molecules in a preparative scale (2-3 mg HLA-DR1) from endosomal compartments by Percoll density-gradient centrifugation. Peptides associated with HLA-DR1 in these intracellular fractions were released, purified by microbore HPLC, characterized by sequencing, and compared with the amino acid composition of peptides derived from MHC class II molecules obtained by solubilization of the plasma membrane. The binding affinity of these MHC fractions was analyzed by our highly sensitive binding assay using different DR1-restricted IM and Ii peptides. The results indicate that (a) intracellular MHC molecules show higher peptide-binding capacity, (b) peptides that are about 18-25 amino acids long need only a core region of 11 amino acids for binding, (c) specific positions of the peptides are important for DR1 binding, (d) most of the naturally processed peptides show a proline at position 2 or 3 that may represent a stop signal for trimming, and (e) Ii peptides are very abundant in DR1 peptide pools derived from intracellular compartments.

Amino Acid Sequence↗

PubMed1992

Self-peptide released from class II HLA-DR1 exhibits a hydrophobic two-residue contact motif.

Peptide fragments of foreign and self-proteins are of great immunologic importance as their binding to major histocompatibility complex (MHC) class I or II molecules makes an interaction with a corresponding T cell receptor possible. Recently, allele-specific peptide sequence motifs proved to be responsible for MHC binding, no matter whether self- or non-self-antigens were involved. Up to now, all investigated human class II-associated peptides were derived from foreign antigenic proteins. Therefore, we undertook sequence and binding analyses with a 16-mer self-peptide (SP3) that has been eluted from HLA-DR1. Here we demonstrate, by synthetic polyalanine-based 13-mer analogues of SP3, that two bulky hydrophobic anchor residues with relative spacing i, i + 8 are sufficient for high affinity binding. This is consistent with the hydrophobic i, i + 8 binding pattern recently found for DR-restricted T cell epitopes. Nevertheless, highly helical alanine-based design peptides with anchor spacing i, i + 9 exhibit maximal affinity, whereas replacement of alanine by helix destabilizing proline abrogates binding. Thus, a two-residue contact motif is the common minimal requirement of self- and foreign peptides for high affinity anchoring to HLA-DR1. In contrast to class I, the anchor spacing of DR1-associated peptides seems to bear some variability due to conformational diversity.

Amino Acid Sequence↗

PubMed1991

Self and foreign peptides interact with intact and disassembled MHC class II antigen HLA-DR via tryptophan pockets.

The acid release of endogenous peptides from immunoaffinity-pure human major histocompatibility complex (MHC) class II proteins HLA-DR1 is accompanied by an 18% decrease in intrinsic tryptophan fluorescence. The effect is totally reversible upon readdition of an autologous endogenous peptide fraction. High-performance size-exclusion chromatographic (HPSEC) binding and release studies with a nonfluorescent HLA-DR1-restricted influenza matrix peptide IM(18-29) prove the fact that Trp residues of the HLA protein change their fluorescence intensities. Since the far-UV circular dichroism spectra of HLA molecules before and after peptide release, DR1[NAT] and DR1[REL], show very small differences, we can rule out the breakdown of secondary structural elements under release conditions, although DR[REL] consists of disassembled alpha- and beta-subunits, as evidenced by HPSEC. Quenching of DR1[NAT] and DR1[REL] using the neutral quencher acrylamide results in a 20% increase in total accessibility of the nine-residue Trp population whereas quenching by iodide yields only a 5% increase. Both results taken together tell us that two Trp residues, preferentially ones located in apolar pockets, become accessible upon the release of peptides. The significantly smaller fluorescence enhancement upon binding IM(18-29) of DR3[REL], exclusively lacking Trp-9(beta 1), and the missing tendency to reassemble under the influence of IM(18-29) compared to DR1[REL] suggest an important role for position 9(beta 1). The region around Trp-43(alpha 1) should be responsible for the binding of IM(18-29) to the alpha-subunits of DR1 and DR3, respectively, as verified by fluorometric HPSEC and SDS-PAGE. Obviously, our findings are in total agreement with the hypothetical MHC class II model, whereafter Trp-9(beta 1) and Trp-43(alpha 1) besides Trp-61(beta 1) are constituents of the binding groove of DR1. Extending the homology to MHC class I products, we postulate the existence of three hydrophobic pockets in the binding site of DR1 with the cited Trp residues being juxtaposed to contacting apolar peptide side chains in HLA-peptide complexes. According to the deduced two-residue-contact model the minimal consensus motif for DR1-restricted peptide antigens consists of two hydrophobic residues lying 14-16 A apart in the bound state of the peptide.

Amino Acid Sequence↗

PubMed1991

Non-radioactive detection of MHC class II-peptide antigen complexes in the sub-picomole range by high-performance size-exclusion chromatography with fluorescence detection.

In order to avoid chemical or structural modification of T-cell epitopes by labelling, a high-performance size-exclusion chromatographic fluorescence binding assay was developed, based on the intrinsic Trp fluorescence of major histocompatibility complex (MHC) proteins. The increase in Trp fluorescence intensity of the isolated human MHC product HLA-DR 1 on complex formation with unlabelled influenza matrix peptide[18-29] (IM[18-29]) was examined. Binding of IM[18-29] to the heterodimeric form of HLA-DR 1 (Kd = 4.8 mM) and to the disassembled alpha-and beta-subunits (Kd = 9.2 mM) could be demonstrated. In addition, the assay showed the peptide-induced formation of a dimeric conformer of HLA-DR 1, the nature of which is still undefined. Detection of HLA-DR 1 subunit-peptide complexes was possible in amounts of 25 ng in 10 microliter (80 fmol/microliter). The technique proved to be reproducible and less time consuming than common methods that need fluorescence or radioactive labelling.

Chromatography, Gel↗

PubMed1983

[Articular ligament rupture of the ankle joint].

We report about 142 cases of acute injuries of the fibular ligament lesions of the ankle joint treated by surgery. In 2.1% no rupture was found. A follow-up examination and X-ray examination was done after several month for 75 of these patients, and a good result without any sign of instability of the ankle joint was achieved in 84% to 86.7%. Complications were found in 2.1% (secondary healing, no further complications). In order to these results we recommend the treatment of the fibular ligament lesion by surgery.

Adolescent↗

PubMed1977

[Optimum delivery methods in breech presentation].

In 581 cases of breech presentation during the years 1966--1974 in 90% of cases delivery was possible vaginally. We prefered the method of Lövset und Veit-Smellie. Casarian section was performed in 9,5% of the cases, the mortality of the newborns was 14%. Without the premature newborns (less than 1000 gr), the cases of intrauterine deaths and not viable children with malformations the mortality was 5,6%. The perinatal mortality of the children or more than 2500 gr was merely 0,69%. The general enlargement of the indication for Caesarian section is not recommended.

Adult↗

PubMed1976

[On the optimal method of delivery in breech presentation (author's transl)].

Between 1966 and 1974, 90% of 581 single breech presentations were delivered vaginally. The incidence of Caesarean Section was 9.5%. The overall perinatal mortality was 14%. After elimination of premature deliveries under 1000 grams, stillbirth prior to labour and non-viable anomalies, the perinatal mortality was 5.6%. The perinatal mortality in infants over 2500 grams was 0.69%.

Birth Weight↗